Assay Development for the Identification of NEDD8- activating Enzyme Inhibitors

鉴定 NEDD8 激活酶抑制剂的检测方法开发

• 批准号: 8977494
• 负责人: Matthew Petroski
• 金额: $ 44.61万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-03 至 2017-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8977494
• 关键词: Affinity; Basic Science; Binding; Biochemical; Biological; Biological Assay; Cancer Patient; Cell physiology; Cells; Chemicals; Chronic; Clinical; Clinical Data; Clinical Trials; Cullin Proteins; Data; Development; Disease; Enzyme Inhibition; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzyme Kinetics; Enzymes; Equilibrium; Etiology; Experimental Neoplasms; Health; Human; Inflammation; Libraries; Ligands; Lysine; Malignant Neoplasms; Measures; Mediating; Mutation; Normal Cell; Pharmaceutical Preparations; Phase; Post-Translational Protein Processing; Property; Proteins; Regulation; Research; Resistance; Role; Signal Pathway; Specificity; System; Testing; Therapeutic; Therapeutic Agents; Translational Research; Ubiquitin; Ubiquitin Like Proteins; Work; adenylate; antitumor effect; assay development; base; cancer cell; cancer therapy; enzyme activity; high throughput screening; improved; in vivo; inhibitor/antagonist; innovation; insight; interest; mimetics; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; pre-clinical; preclinical study; response; screening; small molecule; small molecule inhibitor; small molecule libraries; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): The ubiquitin-like protein NEDD8 functions to regulate the activation of cullin-RING ubiquitin ligases (CRLs). Although CRLs have well-established roles in many fundamental aspects of cellular physiology, recent pre- clinical studies have found that cancer cells are more reliant on NEDD8 for proliferation and survival than normal cells. These observations have invigorated interest in devising strategies to pharmacologically inhibit NEDD8 to treat cancer and other diseases. The NEDD8-activating enzyme (NAE) represents the most attractive target of the NEDD8 system as it possess a catalytic pocket suitable for binding small molecules and undergoes structural rearrangements important for its activities. MLN4924, a mechanism-based inhibitor that generates a NEDD8-adenylate mimetic in the NAE catalytic pocket, represents the only NEDD8 system inhibitor and is currently being evaluated on cancer patients. We and other have found that cancer cells and experimental tumors rapidly lose sensitivity to MLN4924 through treatment-emergent mutations in NAE that impact inhibitor binding by altering the enzyme's biochemical properties. Although the implications of these on MLN4924 therapy are currently unknown, they suggest cancer cells tolerate considerable variability in NAE function. These mutations render cells broadly resistant to molecules targeting the enzyme's catalytic pocket. This project, submitted in response to PAR-13-364 Development of Assays for High-Throughput Screening for Use in Probe and Pre-therapeutic Discovery, seeks to develop highly innovative biophysical and cell-based assays focused on the NEDD8 system and to use these in pilot screens to identify proof-of-concept probes. Through our preliminary studies, we have devised a homogeneous biophysical assay that measures ligand- dependent changes in NAE thermal stability. We hypothesize that this assay will allow for different classes of equilibrium-binding reversible inhibitors to be identifie by screening chemically diverse small molecule libraries. To test this hypothesis and achieve the overall objective of the research, three Specific Aims are proposed: 1) to establish and validate biophysical assays that measure NAE thermal stability and supporting specificity assays; 2) to develop cell-based assays that measure biological effects of NAE inhibition; and 3) to conduct focused high-throughput screens with selected libraries, verify hits, and determine hit potency and selectivity. IMPACT: This work is highly innovative with translational relevance based on the extraordinary opportunity it provides to develop new research tools and therapeutic approaches to inhibit NAE and the NEDD8 system for the treatment of cancers and other diseases. Through our efforts focused on generating novel assays and chemical probes, these studies are expected to provide important new insight into the NEDD8 system and other ubiquitin and ubiquitin-like protein modification systems to improve human health.

描述（由申请人提供）：类似泛素的蛋白NEDD8功能，以调节Cullin环泛素连接酶（CRLS）的激活。尽管CRL在细胞生理学的许多基本方面具有完善的作用，但最近的临床研究发现，癌细胞比正常细胞更依赖于NEDD8进行增殖和存活。这些观察结果激发了人们对制定策略以抑制NEDD8治疗癌症和其他疾病的兴趣。 NEDD8激活酶（NAE）代表了NEDD8系统的最具吸引力的目标，因为它具有适合结合小分子的催化口袋，并且经历了对其活性很重要的结构重排。 MLN4924是一种基于机制的抑制剂，在NAE催化口袋中产生NEDD8-辅助酸酯模拟物，代表了唯一的NEDD8系统抑制剂，目前正在对癌症患者进行评估。我们和其他人发现，癌细胞和实验性肿瘤通过NAE的治疗突变突变迅速失去对MLN4924的敏感性，这些突变通过改变酶的生化特性而影响抑制剂结合。尽管目前这些对MLN4924治疗的含义尚不清楚，但它们表明癌细胞耐受NAE功能的差异很大。这些突变使细胞对靶向催化口袋的分子具有广泛的抗性。该项目是针对Par-13-364的开发，用于开发用于用于探针和治疗前发现的高通量筛选的测定法，旨在开发针对NEDD8系统的高度创新性生物物理和基于细胞的测定，并将其用于NEDD8系统并将其用于试验筛查以识别验证探针。通过我们的初步研究，我们设计了一种均匀的生物物理测定法，该测定法测量了NAE热稳定性的配体依赖性变化。我们假设该测定法可以通过筛选化学多样的小分子文库来识别不同类别的平衡结合可逆抑制剂。为了检验这一假设并实现研究的整体目标，提出了三个具体目标：1）建立和验证生物物理测定法，以测量NAE热稳定性和支持特异性测定； 2）开发基于细胞的测定，以测量NAE抑制的生物学作用； 3）用选定的库进行聚焦的高通量屏幕，验证命中率，并确定命中效率和选择性。影响：这项工作具有高度创新性，基于它提供的非凡机会，以开发新的研究工具和治疗方法来抑制NAE和NEDD8系统来治疗癌症和其他疾病。通过我们的努力着重于生成新颖的测定法和化学探针，这些研究有望为NEDD8系统以及其他泛素和泛素样蛋白质修饰系统提供重要的新见解，以改善人类健康。